Fluorescent nanoscopy method

ABSTRACT

A method for analysis of an object dyed with fluorescent coloring agents. Separately fluorescing visible molecules or nanoparticles are periodically formed in different object parts, the laser produces the oscillation thereof which is sufficient for recording the non-overlapping images of the molecules or nanoparticles and for decoloring already recorded fluorescent molecules, wherein tens of thousands of pictures of recorded individual molecule or nanoparticle images, in the form of stains having a diameter on the order of a fluorescent light wavelength multiplied by a microscope amplification, are processed by a computer for searching the coordinates of the stain centers and building the object image according to millions of calculated stain center co-ordinates corresponding to the co-ordinates of the individual fluorescent molecules or nanoparticles. Two-dimensional and three-dimensional images are provided for proteins, nucleic acids and lipids with different coloring agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/891,420, filed on 27 Sep. 2010, and issued as U.S. Pat. No.8,110,405, which is a continuation of U.S. patent application Ser. No.11/920,661, filed on 19 Nov. 2007, and issued as U.S. Pat. No.7,803,634, which is a National Phase Patent Application ofPCT/RU2006/000231. The co-pending parent application is herebyincorporated by reference herein in its entirety and is made a parthereof, including but not limited to those portions which specificallyappear hereinafter.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to equipment for scientific research, and lensfluorescence microscopes in particular, that are used for obtainingimages of fluorescent objects immobilized on a glass. This inventionalso relates to a computerized fluorescent-microscopically method ofreconstructing images of objects with resolution up to severalnanometers (nm).

2. Discussion of Related Art

There are known optical microscopes which can create zoomed images of anobject with object lenses that can show two spots on an objectseparately only when the distance between them is more than so calleddiffraction limit. This limit can be calculated using following formula:r=0.61λ/A (1), where λ is light wavelength for light collected by objectlens with aperture A=n*sin(φ), n is a refraction index of substancewhich surrounds object spots, φ is an angle between object lens axis andextreme rays which fall into object lens and are detected in detector.Now, different types of devices are used for fluorescent microscopy byobject lenses. Powerful arc-lamps, incandescent lamps, laser or sunlight can be light sources for the microscope. Fluorescence starts inall dye molecules are present in a lighted area. The area is lightedthrough the object lens using a light-dividing dichroic mirror that letsexciting light to fall on the object and reflects fluorescence light tothe detector. The second type of lighting occurs by sending laser lightfrom the side and lights the object all the way down or through anobject glass at a total internal reflection angle. In this case, lightreaches a depth of only 0.3 of light wavelength from border betweenglass and an object which has a refraction index lower than glass.Object fluorescent light is collected by an object lens and sends anobject image for visual observation and registration by aphotomultiplier, a photographic tape or a digital video-camera. The maindisadvantage of all existing lens microscopes is that they have a limitof resolution for two neighboring spots and the limit can be calculatedaccording to the above formula (1).

Recently, microscopy using super lenses made from silver film has beendeveloped. A film thickness less than 50 nm can assure resolution of twospots on a distance of approximately 50 nm from each other. (N. Fang andX. Zhang, Imaging properties of a metamaterial superlens, 2003, App physLet v. 82, 2, 161-163; Nicholas Fang, Zhaowei Liu, Ta-Jen Yen, and XiangZhang Regenerating evanescent waves from a silver superlens, 2003,OPTICS EXPRESS, VoI. 11, No. 7, 682-687). Use of such microscopes onbiological objects is unclear. The present microscope model has aresolution several times lower than a resolution of the device that wepropose.

There are devices with a maximum resolution better than 1 nm, forexample, electronic, tunnel, and atomic-force microscopes that have notonly real advantages but also serious disadvantages, such as: complexityand expensiveness of their design and work with objects; lack ofopportunity to receive a color image for distinguishing molecules ofdifferent types; objects that usually should be dried and treated withsubstances which change the mutual layout of different parts of theobject. Atomic and tunnel microscopes also do not detect structuresinside the object; only one spot can be detected at a time and ascanning speed does not overcome 1 square micron per min; and an end ofthe needle easily becomes dirty and does not then reach an objectsurface.

There is also a device, where object fluorescence is excited by laserthrough a pinhole on the tip of a glass fiber. The fiber is moved bydrives in three directions to position an end of the fiber nearlight-reflecting, light-diffusing or covered with fluorescent moleculessurface. This type of microscope does not use lenses and permitsobtaining images with resolution ten times better than the resolution ofcommon optical microscopes. These results can be reached only when thepinhole on the tip of glass fiber is much less in diameter than in lightwavelength. The light comes onto an object with a depth much shorterthan a light wavelength. Practically all light returns back into theglass fiber, except that part which was captured by objects from outsideof the hole. Fluorescence, light-diffusion, and reflected lightstrength, captured in the object near the hole, are measured by aphotomultiplier. The image of an object surface is reconstructed by acomputer, which gathers information about the strength of measured lightand data on an end of glass fiber coordinates. The main disadvantages ofthis system are: a need to use expensive high-precision and fast-actingmechanic units, responsible for moving of glass fiber against object;production of glass fiber with an end hole less than 50 nm in diameteris very expensive, complex and difficult for duplicating; the hole iseasily dirtied and is not able to then reach an object surface; only aninsignificant part of light can leave the fiber through the hole with adiameter less than light wavelength; increases of light intensity leadsto overheating and destruction of the fiber end; it is impossible todetect fluorescence in the areas of the object which are not accessibleby glass fiber; and only one spot can be detected at a time, and surfacescanning speed does not exceed 1 square micron per minute.

One more new microscope type scans an object surface with several lightbeams simultaneously. National Institute of Standards and Technology(NIST) issued a grant for 5 years research work on creation of thismicroscope(http://www.betterhumans.com/News/news.aspx?articleID=2005-02-11-4,“Optical Microscopes Enter the Nano Age. Hybrid system being developedto image and measure features smaller than the wavelength of visiblelight”.) The article states that a 40 nm nanoparticle can bedistinguished using this method. There are no indications of authorsbeing successful in distinguishing two separate particles located on adistance less than 40 nm from each other. It is not clear from presenteddrawings and explanations how this method will allow distinguishing twoparticles on distance less than r<0.61λ/A between them and the suggesteddevice will likely not reach resolution of object details, located on adistance much shorter than light wavelength.

One method of using common fluorescent microscope (Erwen, A Sharonov, JH Ferris, R M Hochstrasser: Direct visualization of nanopatterns bysingle-molecule imaging. App Phys Let 2005, 86: 043102) can be thoughtof as relevant to this invention. The main idea of this method is that asample, a light film of polymer with free open spherical cells 1 micronin diameter, is dyed with very low concentration of fluorescent peptide.Such concentration allows to observe separate peptide molecules whichcan migrate in Brownian motion inside the hollow of spheres. The sampleis lighted through an object glass by a laser beam. A lightning angle isequal to a total internal reflection angle. A laser beam excitesfluorescence in a 150-200 micron layer of the sample near the glass. Alocation of several tens of molecules of fluorescing peptide, each ofwhich was dyed by simultaneously fluorescing molecules, was detected bya high-sensitivity video camera (Roper Scientific, Cascade 512F withelectrons multiplier built in CCD) in 500 sequential frames. Each framewas recorded to computer memory. Each image contained many spots ofapproximately 0.5 microns in diameter multiplied on a system zoom value.All these images were added to each other and a resultant image had aresolution not exceeding a resolution of a common fluorescentmicroscope. Main disadvantages of this system are: there is noinformation in the article about opportunity to calculate a location ofdetected spot centers and to generate an image on the basis of thesespots with resolution higher than a diffraction limit (see formula 1);approaches to selective dyeing of object structures are not described inthe article; and there is no description of solving that after dyeing,substances lose color in the process of detecting non fluorescingpeptide molecules staying in an observation area. This will make thesolution more thick and will not allow replacing such molecules with newfluorescing ones. This does not allow obtaining larger quantity offrames than 500. These large quantities are needed to receive image withresolution higher than allowed by a diffraction limit (see formula 1).

SUMMARY OF THE INVENTION

Researching of biological objects, for example muscles, includes thefact that many different types of molecules are located much closer thanthe resolution of common optical microscopes which create a zoomed imageof the object by an object lens in a plane of a recorder, eye, photo orvideo camera. The diffraction limit of microscope resolution (formula 1)limits the resolution in microscopes with simultaneous observation ofall spots in the observation area and in microscopes with sequentialobservation of all spots of an object by a light ray being focused onone spot (confocal and other types of scanning lens microscopes). Thatis why it is good if all the advantages of optical microscopes and usedmethods of selective dyeing of different types of molecules in a visionarea are united with such improvement of resolution, which would allowseparate observation of molecules located less than 10-20 nm from eachother.

This invention provides methods of dyeing objects, preparing objects forresearch, computer analysis of results. This procedure will allowreceiving an object image with resolution higher than 20 nm. This willturn a fluorescent microscope into a “nanoscope”. This task is solved bymaking multiple pictures of low dyed objects, (all fluorescing moleculesin the objects are seen separately as spots with a diameter of2r=1.22λ/A, having different location on tens of thousands of sequentialframes). Further, all these frames are used to calculate locations ofcenters of all detected spots (the locations correspond with actualcoordinates of fluorescing molecules). Then 3-D reconstruction of alldye molecule locations is performed. Resolution is comparable with afluorescing molecule size. Different structures of the object can bedyed in different colors. Fluorescent nanoscopes can be based onstandard modules, used in fluorescent microscopy. This inventioncomprises various modules: an optical system for visual observation andtransmitting of object image to digital video camera. The video camerasshould be able to detect and digitize images of separate fluorescingmolecules and nanoparticles with low background noise level. A secondmodule of the system is a computer for recording and analyzing images. Athird component is a sample holder, located opposite to objective lens.A fourth component is a set of changeable suppressing color filters forpicking up light of sample fluorescence. The nanoscope should beequipped with two light sources installed aside from a sample holder. Aninstallation angle should assure lighting of an entire slice of thesample on full depth or in a layer less than 150 nm near the glass.Fluorescent molecules in this layer can absorb energy of light waves,flowing through the border when lighted on a total internal reflectionangle. Observation objects should be preliminary dyed in a solution witha saturating concentration of caged dye which starts fluorescing onlywhen a UV light pulse separates blocking fluorescence groups from dyemolecules. Superfluous dye should be carefully washed out. It is veryhard to assure stability of molecular structures for a long time farfrom covering a glass and a prism glass holding the object. This is truefor objects observed exactly in a fresh flushing solution. That is whythe best resolution of a nanoscope can be received only for those layersof the object which are located on the distance not more than 150 nmfrom the prism glass. This is the depth which light reaches when fallingon the boundary surface at a total internal reflection angle. In case ofobserving a dead preserved object it is usually possible to observe theobject during many hours. Different structures, located near a baseglass are almost an absolute immovable. They keep their location becauseof multiple stable mutual connections and connections with the baseglass. They are immovable in spite of the fact that nearly 80% of volumeis occupied with an aqueous solution of different salts. Gaps on glassedges should be hermetically sealed (for example, with paraffin) toavoid drying of the solution.

3-D nanoscopy requires total immobility of the object and its partsagainst an objective lens. That is why the object, after being dyed withcaged dye and flushed, should be treated with dehydrating solutions andplaced into a polymer non-fluorescent substance (for example, epoxy).Microscopic sections of the object of several microns thickness can beused for 3-D reconstruction of the object with 10-20 nm resolution inall three dimensions. Such an object contained in a solid substance canbe lighted as it is done in common fluorescent microscopy. Only animprovement of an additional flash-lamp should be used in order to turnseveral hundred non-fluorescing molecules into fluorescing in eachframe. For both, object in liquid and object in polymer film, a UVflash-lamp with λ<360 nm periodically lights the object, dyed withnon-fluorescing dye, and each time turns several hundred (and even athousand) of non-fluorescing molecules into fluorescing ones byphotolysis of special chemical groups blocking fluorescence. The laserconstantly lights the object and excites fluorescence of newly formedfluorescent molecules with such intensity, that each of them will sendseveral tens of thousands of light quanta in part of a second. Moleculeswill lose color after registration of its fluorescence on a digitizedframe with low background noise level. Cycling of fluorescent moleculesgeneration, their excitation, registration and losing color can berepeated tens of thousands of times. Each time new fluorescencemolecules will be generated, detected and will lose color. Not convertednon-fluorescent molecules will not lose color because they do not absorblaser light. Tens of thousands of frames can be detected using thismethod. Hundreds and even thousands of fluorescing molecules can bedetected on each frame. It will allow calculation of locations of alltens of millions of fluorescing molecules covering surfaces of allstructures, located in a field of vision. Dyes with different activegroups can be used for dyeing structures of different nature indifferent colors. Active groups can link to either proteins, eithernucleic acids, either fats, etc. (one suggested method name is colornanoscopy).

The second modification of a nanoscopy method is based on the fact thatthe Brownian motion of very bright fluorescing and color loss-resistantnanoparticles is possible inside most parts of volume of a biologicalsample with penetrated membranes, filled with salt solution.Nanoparticles are presented by phycobiliprotein molecules or fluorescingmicro spheres 10-40 nm in diameter. This motion can be regulated byelectrophoresis with current, provided by several pairs of interlacingelectrodes. Current directs the particles motion in differentdirections, so they scan all accessible volume. If several hundreds ofthe same, or replaced with those which are out of lighted area,fluorescing particles that are moving on the distance more than 1-2microns from each other are detected in each frame, then it is possibleto calculate their location with several nm accuracy, as coordinates ofcenters of corresponding spots. Coordinates from all frames can be usedfor finding out all locations where particles can appear over a longtime period. It indicates all volume that is occupied with liquid. Partof the volume, where particles were not able to be present, can beconsidered to be occupied with dense structures of different origin,peptides, chromosomes, parts of not damaged membranes, etc. Possiblefluctuations of a particle location can enlarge a spot size a little,but a spot center location can be considered as an averaged coordinateof the particle in the object. It should be noted, that particles withpositive charge, negative charge, and neutral particles can visitdifferent areas of the object because they interact with local chargesof bio-structures, and that is why this method can be useful forstudying surface charges of object structures, such as a suggested nameof the method being monochrome nanoscopy.

Both suggested methods can assure a nanoscope resolution up to severalnm, depending not from resolving power (formula 1), but from intrinsicmobility of structures in an observed part of the object during a longtime period. A second factor is a proportion between noise and signal invideo data. Both factors can be adjusted by various modifications ofpresented methods. For example, some bright shining key fluorescingparticles can be introduced into the object. Then coordinates ofreplacing particles can be calculated taking into account movement in avision area of key particles rigidly connected with the object. Suchapproach allows reaching 2-D distinguishing parts of the object, locatedon the distance of several nm.

It should be noted that some parameters of fluorescing particles images,a diameter of spots and light intensity distribution along a section ofeach spot, can change as particle distances from a focal plane of amicroscope object lens. That is why it is suggested to perform acalculation of these parameters in order to calculate a third coordinateof the shining particle. It is suggested to project an object image ontwo video-cameras. This will improve precision of the third coordinatedefinition. The light rays are divided in two rays after the objectlens. Video-cameras are located so that two neighboring focal planes ofthe object would be projected on them through one object lens. Lightintensity distribution for each particle will be different when detectedby different cameras. It can be scaled depending from the thirdcoordinate and used to create 3-D images of the objects. FIG. 3 shows anillustration of 3-D nanoscopy foundations. A diameter and intensity ofspots from fluorescing particles which are projected on CCD, dependsfrom a distance between the particle and the object lens. Two cameras,focused on different focal planes, show one particle with differentdiameters and a different intensity distribution along diameters. Thediagrams in the upper right corner of the figure illustrate differencesbetween diameters and a distribution of intensity along diametersdepending from cameras and a fluorescing particles location.

A nanoscope in some cases can be helpful in finding centers of spotswhich are created by fluorescing particles in two-three differentwavelengths and same exciting status. It can be done by separating lightdichroic mirrors that pass light with one wavelength to one camera andreflect light with the other wavelength to the other camera. Thus, thisinvention allows to reconstruct simultaneously with a nano resolutionlocation of several different types of object molecules when havingthese molecules dyed with dyes having different emitting wavelengths,and this will allow enlarged quantities of simultaneously seen anddetected separately spots from fluorescing particles due to the factthat they can be seen separately in waves of different lengths, even ifcorresponding spots of different colors are seen interfering.

It can be very useful if described methods of nanoscope work arecombined with an additional changing of caged non-fluorescing moleculesinto fluorescing ones. This can be done by chemical reactions ordifferent physical influences which will separate groups which blockfluorescence. Examples of such reactions are ATP reactions, esteraseinfluence, peroxidation, radioactive transformation, etc. Fluorescingmolecules, created as result of such modifications, can be excited bylaser and detected with video-cameras, used for calculating coordinatesof an object structure surface according to above described methods.Their coordinates can be calculated as centers of appropriate spots onvideo frames. Then their images can be “overlapped” on an earlierreconstructed image of the object. They will show a location of reactionactive groups of the object. Resolution of such image will be betterthan 20 nm.

This invention is described in the attached claims, and can be modifiedwith different methods, not stepping aside from basic ideas of thisinvention.

Operation of the device in nanoscope mode can be altered by using afluorescence microscope in the normal mode. This mode gives opportunityof direct visual observation of the object in situ with commonresolution (formula 1). It also gives an opportunity to detect an imagewith a digital video-camera of very high sensitivity and to transmit itto computer memory. Then these frames will be analyzed to measuregeometric parameters and light intensity in different areas of theframe. This can be used, for example, during bioluminescent andchemoluminescent research.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings show how a fluorescent microscope can be modified toimplement a nanoscopy method according to this invention.

FIG. 1 is a fluorescent microscope-nanoscope equipped with onemonochrome video camera.

FIG. 2 is a fluorescent microscope-nanoscope equipped with twomonochrome video cameras.

FIG. 3 shows an illustration of 3-D nanoscopy foundations.

DETAILED DESCRIPTION OF THE INVENTION

The fluorescent microscope-nanoscope, as it is shown on FIGS. 1 and 2,is equipped with: one (FIG. 1) and two (FIG. 2) monochrome video-cameras(1) with digital output and suppressing color-filters, located oppositeto their sensors (CCD). These color-filters pass only fluorescent lightto cameras. A microscope is also equipped with a light dividingremovable prism (2); object lens (3) with zoom up to 100× and anaperture up to A=1.4. Object (4) is pressed down to glass object holder(5) which is beveled in the form of truncated prism edges. The devicealso has laser (6) with lens system for exciting fluorescence throughprism planes; with a pulse UV-source (7) with a lens system forphotolysis of blocking fluorescence groups, present on dye molecules.Another important part is computer (8) with software for recording andworking with digitized images. This software is also used for control ofa power source, and providing energy for UV pulses and electrophoresisdevice. Eye lens (10) (FIG. 1) is used for visual observation (9). Unitshould be placed into soundproof cabinet, placed on an anti-vibrationtable. The device, providing power to electrophoresis electrodes whichdirect movement of mobile fluorescing nanoparticles inside the object,filled with salt solution, will be described separately.

This is an example of receiving an image with a nanoscope which useshigh sensitivity cameras Cascade 1K, such as produced by Photometrics,or cameras SIS1_t285EM, or produced by Theta-system. SIS1_t285EM camerasare equipped with CCD TC285SPD, produced by Texas Instruments, withelectrons multiplier which is included in CCD crystal. It has a quantumeffectiveness up to 63%, 1004 active horizontal square pixels with sideof 8 microns in 1002 lines on a photosensitive square of approximately8×8 mm. If an object image is projected directly to CCD of avideo-camera by a 100× object lens, then square on the object, lightedwith a laser and its lens system, should be a little bit larger than80×80 microns. Each CCD pixel corresponds with an object square of 80×80nm. Each shining particle or molecule is seen on the object as a spotwith a diameter of 2r=1.22λ/A, which is near 560 nm. It will beprojected to a CCD square with a diameter near 7 pixels. The averagedistance between chaotically spread particles in each frame should bemore than 2000 nm. In this case, practically all of them will be seenseparately. In this case, at least 40×40=1600 spots can besimultaneously projected to CCD in each frame in such a manner, thatthey will be observed separately. Different methods can be used to reacha concentration of simultaneously fluorescing particles. First, it ispossible to dye an object with brightly fluorescing particles in lowconcentration. This will allow them to move in an observation area in aBrownian motion and they can be additionally moved with anelectrophoresis current. Current is provided with several pairs ofelectrodes in such manner that it will direct particles to move indifferent directions. Secondly, the object can be dyed with special dyeslike 5-carboxyfluorescein-bis-(5-carboxymetoxy-2-nitrobenzil) ester,beta-alanin-carboxylamid, suxynimidil ester (CMNB-cagedcarboxyfluorescein, SE), fabricated by Molecular Probes, USA. Afterlighting with a UV flash with a wavelength of 310-365 nm (with apreviously adjusted exposition) approximately 1000-1600 molecules ofthis non-fluorescing dye in an observation area will loose specialgroups which block fluorescence. Such molecules are able to produceseveral thousands quanta of green light when lighted with blue light.After this they will lose color. When an objective lens aperture isA=1.1-1.3, more than 10% of light from each fluorescing particle will betransmitted to a video-camera and participate in formation of the spot,covering near 40 pixels of CDD. Light intensity in the center of thespot is up to 100 quanta. This is enough to receive a video signal withquite a satisfactory proportion of signal and noise (when using theabove mentioned cameras). This proportion will allow calculatingcoordinates of a spot center with resolution higher than 20 nm. This dyedoes not absorb laser light and lose color while groups which blockfluorescence are linked to it. That allows repeating the followingprocess tens of thousands of times: in the beginning CCD detects theimage of the object with residual fluorescence. Then the image isdigitized in a video-camera and is transmitted to computer. Then UVflash creates 1000-1600 fluorescing molecules in a vision area, andlaser excites fluorescence in these molecules. CCD detects images offluorescing molecules and particles, overlapped on residualfluorescence. The image is digitized in the video-camera and istransmitted to a computer where the previous frame with a residualfluorescence is subtracted from it. The received subtracted frame isstored in computer memory for further analysis with the purpose ofcalculating coordinates of a spot center, its averaged diameter, andintensity. Then the object is for some time lighted with laser withoutdetecting its image to maximize color loss of already detectedmolecules. Then the whole cycle is repeated. New fluorescing moleculesare created, detected and lose color in each cycle until coordinates ofall dye molecules are detected. Molecular Probes produce not onlymentioned dye. Dyes can be produced on request. It also produces sets ofreagents for individual creating of such dyes, such as a “caging kit”(D-2516).

Cameras, indicated in the example, allow recording images of separatefluorescing molecules with a frequency of 10 frames per second. Imagesare recorded with satisfactory proportion of signal and noise. It ispossible to detect, for example, 40000 frames during several hours. Eachframe will contain up to 1600 images of molecules. If so, a totalquantity of detected fluorescing molecules' coordinates can reach 64million, an average distance between observed separately molecules canbe only 10 nm. This is tens times better than in any other lensmicroscopes. Frames with molecule images should be saved withoutcompressing or compressed without loosing quality. This should be donefor more exact calculating of molecule coordinates. A total volume ofinformation on all not compressed frames in one experiment can reachtens of gigabytes. This will not create a problem taking into accountmodern hard disk drives volume. Literature sources provide informationon various algorithms of calculating spot centers. Computer software hasbeen created to perform such calculations and reconstruction of a fullimage based on a table of calculated coordinates of spot centers.

1. A method for 3D microscopy of objects, comprising: introducing afluorescent agent within an object; exciting the fluorescent agent;capturing with at least one camera and a data processor a plurality ofimage frames including separately observed fluorescing spots produced bythe excited fluorescent agent; saving the captured image frames to arecordable medium associated with the data processor; obtaining morethan one intensity distribution for each fluorescing spot from differentfocal planes with a light dividing prism and the at least one camera;and constructing a three-dimensional image of the object usingdifferences between the light intensity distributions of each spotdetected between the different focal planes.
 2. The method of claim 1,further comprising introducing the fluorescent agent at a lowconcentration that allows the fluorescent agent to be detectableseparately as spots when agent are excited.
 3. The method of claim 1,wherein the light dividing prism and one or more cameras are positionedso neighboring focal planes of the object are projected through oneobject lens.
 4. The method of claim 1, wherein the fluorescing spotscorrespond with lateral coordinates of the fluorescent agent.
 5. Themethod of claim 1, wherein the fluorescent agent comprises fluorescentnanoparticles or fluorescent molecules.
 6. The method of claim 1,wherein reconstructing the three-dimensional image comprises calculatingan axial coordinate of each spot with an accuracy up to several nm. 7.The method of claim 1, wherein the fluorescent agent is a dye and theobject is a dyed object, and further comprising: generating fluorescentmolecules by converting non-fluorescent dye molecules into fluorescentdye molecules; exciting the fluorescent molecules of the dyed object,wherein each of the excited fluorescent molecules has a color; andregistering each of the excited fluorescent molecules as the separatespot; and losing the color of the fluorescent molecules.
 8. The methodof claim 1, further comprising: introducing fluorescing nanoparticles asthe fluorescent agent; registering a motion of the fluorescingnanoparticles resistant to fluorescent color loss within a portion of asample filled with solution, the motion allowing a scan of accessiblevolume of the sample and to indicate volume that is filled with liquidand volume that is occupied with dense structures; and calculating alocation of the nanoparticles from corresponding spots from all frames.9. The method of claim 1, further comprising: repeating a cycle of theexciting and registering steps a plurality of times; and constructing athree-dimensional image of the object using differences between thelight intensity distribution for each spot detected between thedifferent focal planes over the cycle.
 10. The method of claim 1,further comprising: introducing fluorescing nanoparticles as thefluorescent agent; registering a motion of the fluorescing nanoparticlesresistant to fluorescent color loss within a portion of a sample filledwith solution, the motion allowing a scan of accessible volume of thesample and to indicate volume that is filled with liquid and volume thatis occupied with structures having the same local charge as thefluorescent agent; and calculating a location of the nanoparticles fromcorresponding spots from all frames.
 11. The method of claim 1, furthercomprising determining lateral coordinates of the spots from the lightintensity distributions of the plurality of focal planes.
 12. The methodof claim 1, wherein reconstructing the three-dimensional image comprisescalculating an axial coordinate of each fluorescent agent spot.